Endonuclase-assisted isothermal amplification using contamination-free reagents

ABSTRACT

Disclosed are methods and kits for endonuclease-assisted DNA amplification reaction using decontaminated primer solutions that are pre-treated with a nuclease. Nucleic acid amplification assays that employ nuclease-resistant, inosine-containing primers, endonuclease V enzymes to introduce a nick into a target DNA comprising at least one inosine, and a DNA polymerase to generate amplicons of a target DNA are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 13/330,745, filed on Dec. 20, 2011, which is a divisional ofU.S. patent application Ser. No. 11/621,703, filed on Jan. 10, 2007,U.S. Pat. No. 8,202,972 B2, both entitled ISOTHERMAL DNA AMPLIFICATION.

FIELD OF INVENTION

The invention generally relates to nucleic acid synthesis methods andagents that employ at least one exonuclease-resistant,inosine-containing primer, at lease one endonuclease that is capable ofintroducing a nick in a double-stranded DNA sequence comprising aninosine residue at a residue 3′ to the inosine residue, and at least onestrand displacement DNA polymerase. It further relates to improved DNAamplification methods wherein the primer solution comprising theexonuclease-resistant, inosine-containing primer, which may furtherinclude reaction buffer and certain accessory protein(s) are pre-treatedwith an exonuclease to remove any contaminating nucleic acids beforegenerating the DNA amplification reaction mixture.

BACKGROUND

DNA amplification is a process of copying a single or double-strandedtarget DNA to generate multiple copies of the target DNA. Since DNAstrands are antiparallel and complementary, each strand may serve as atemplate (template strand) for the production of an opposite strand(complementary strand) by a DNA polymerase. The template strand ispreserved as a whole or as a truncated portion and the complementarystrand is assembled from nucleoside triphosphates. A variety ofefficient nucleic acid amplification techniques are currently availablesuch as polymerase chain reaction (PCR), ligase chain reaction (LCR),self-sustained sequence replication (3SR), nucleic acid sequence basedamplification (NASBA), strand displacement amplification (SDA), multipledisplacement amplification (MDA), or rolling circle amplification (RCA).Many of these techniques generate a large number of amplified productsin a short span of time. For example, in a PCR, a target DNA, a pair ofprimers and a DNA polymerase are combined and subjected to repeatedtemperature changes that permit melting, annealing, and elongation stepsto result in an exponential amplification of the starting target DNA.

DNA amplification often suffers from high background signals due tonon-target or non-specific amplification reactions yielding undesired,false amplification products. For example, nucleic acid amplificationreactions may get contaminated with unwanted nucleic acids (e.g.,nucleic acids other than the target nucleic acid) in various waysyielding non-target amplification products. Contamination may arise fromcarry-over amplification products (amplicons) of previous amplificationreactions, from the site from which the sample for amplification iscollected, by exogenous DNA in the laboratory environment, or fromreagents or reagent solutions used for amplification reaction.Non-specific amplification may result from various primer gymnasticssuch as nucleic acid template-independent primer-primer interactions.For example, primers may form primer-dimer structures by intra- orinter-strand primer annealing (via intra molecular or inter molecularhybridizations), and may get amplified, and may sometimes predominate,inhibit, or mask the amplification of a target DNA sequence. Suchbackground amplification reactions become even more problematic wherethe target nucleic acid to be amplified is available only in limitedquantities (e.g., whole-genome amplification from a single target DNAmolecule). Due to higher amplification efficiencies of the DNAamplification techniques, even the slightest contamination of thereagents or reagent solutions employed in the amplification reactionswith an undesired nucleic acid molecule may result in a huge amount offalse or undesired amplification products. If such amplificationtechnologies are used for diagnostic applications, they would likelyresult in a false-positive diagnosis.

Various pre-amplification sterilization procedures have been developedto minimize these non-target or non-specific amplification reactions.For example, deoxythymidine triphosphate (dTTP) is substituted bydeoxyuridine triphosphate (dUTP) in PCR amplifications to make PCRproducts distinguishable from template DNA. Use of uracil-N-glycosylaseenzyme (UNG) in a pre-amplification step cleaves the carry-overamplicons at the incorporated uracil residues. In amplificationreactions using the same primers and the same target sequences,enzymatic removal of amplicons from previous similar amplificationreactions has also been reported. These methods take advantage of thefact that the contaminant amplicon carries its primer sites at or nearthe ends of the molecule whereas virtually all other template DNAmolecules not arising themselves from a previous PCR reaction, do nothave their primer sites so located. Single strand-specific exonucleasehas been used for amplicon de-contamination during SDA reaction whereineither (or both) the target nucleic acid or the amplicons are in singlestranded form. In such methods, even though both the target andamplicons are attacked, due to the short length of amplicons (25-2,000nucleotides) and their lack of secondary structures, the amplicons arepreferentially cleaved. Selectively activatable enzymes such asmicrococcal nuclease and of DNA-binding agents have also been employedto de-contaminate the reagent solution. Enzymatic, physical or chemicalpre-treatment of the sample has also been employed to remove orinactivate a contaminating DNA that is originating from the site fromwhere the sample is collected.

Apart from amplicon carry-over, reagents and reagent solutions, commonlyused to amplify nucleic acids, may also contain unwanted nucleic acidcontaminants that could potentially interfere with standard nucleic acidamplification protocols and procedures. Contaminating DNA may be muchlonger than that of a primer or an amplicon and specific informationabout the contaminating DNA may often be minimal. So, there exists aneed to de-contaminate the reagents and the reagent solutions used foramplification reactions. As noted, the invention relates generally tothe use of exonuclease-resistant, inosine-containing primers forendonuclease-assisted DNA amplification (referred as “Ping Pong”amplification) as well as the pre-amplification treatment ofamplification solutions containing the exonuclease-resistant primerswith an exonuclease to remove contaminating nucleic acids prior to thePing-Pong amplification reaction.

BRIEF DESCRIPTION

In some embodiments, a nucleic acid amplification method is used thatcomprises the steps of providing a target DNA and a primer solutioncomprising at least one exonuclease-resistant, inosine-contaning primer;generating a DNA amplification reaction mixture by mixing together thetarget DNA, the primer solution, at least one 5′→3′exonuclease-deficient DNA polymerase having strand displacementactivity, and at least one endonuclease that is capable of nicking aninosine-containing strand of a double stranded DNA at a residue 3′ to aninosine residue; and amplifying at least one portion of the target DNAusing the DNA amplification reaction to produce at least one amplicon.Before generating the DNA amplification mixture, the primer solution,which may further include other nucleic acid amplification reagents(e.g., dNTPs, buffers, accessory proteins) may be decontaminated bytreating with at least one exonuclease to remove any contaminatingnucleic acid.

In some embodiments a method for producing at least one amplicon basedon a target DNA is provided, wherein the method comprises the steps of(a) providing the target DNA; (b) providing a primer solution consistingessentially of an exonuclease-resistant, inosine-containing primer; (c)treating the primer solution with an exonuclease(s) to remove anycontaminating nucleic acids from the primer solution; (d) inactivatingthe exonuclease(s) in the primer solution after the decontamination step(c); (e) generating a DNA amplification reaction mixture by mixingtogether the target DNA, the decontaminated primer solution, at leastone 5′→3′ exonuclease-deficient DNA polymerase having stranddisplacement activity, and at least one endonuclease that is capable ofnicking a DNA at a residue 3′ to an inosine residue; and (f) amplifyingat least one portion of the target DNA using the DNA amplificationreaction mixture of step (e) to produce the amplicon. The primersolution may further comprise other nucleic acid amplification reagentssuch as dNTPs, accessory protein(s), and amplification buffers.

An embodiment of kit comprises at least one exonuclease-resistant,inosine-containing primer, at least one 5′→3′ exonuclease-deficient DNApolymerase having strand displacement activity, and at least oneendonuclease that is capable of nicking an inosine-containing strand ofa double stranded DNA at a residue 3′ to the inosine residue.

DRAWINGS

These and other features, aspects and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying figures.

FIG. 1 illustrates a schematic representation of an embodiment ofendonuclease-assisted nucleic acid amplification reaction.

FIG. 2 illustrates a schematic representation of an embodiment ofendonuclease-assisted nucleic acid amplification reaction using anexonuclease-resistant, inosine-containing primer.

FIG. 3 illustrates an example of the effect of exonuclease-resistant,inosine-containing primer on endonuclease-assisted template DNAamplification.

FIG. 4 illustrates an example of the effect of the removal ofcontaminating nucleic acids by exonuclease treatment of amplificationreagents prior to endonuclease-assisted template DNA amplification.

DETAILED DESCRIPTION

To more clearly and concisely describe and point out the subject matterof the claimed invention, the following definitions are provided forspecific terms, which are used in the following description and theappended claims.

As used herein, the term “target DNA” refers to a DNA sequence of eithernatural or synthetic origin that is desired to be amplified in a DNAamplification reaction. The target DNA acts as a template in the nucleicacid amplification reaction. In a DNA amplification reaction, either aportion of a target DNA or the entire region of a target DNA may getamplified by a DNA polymerase to produce one or more amplificationproducts (amplicons). The target DNA may be obtained from a biologicalsample (i.e., a sample obtained from a biological subject) in vivo or invitro. The target DNA may be obtained from, but are not limited to, bodyfluid (e.g., blood, blood plasma, serum, or urine), organs, tissues,fractions and sections (e.g., sectional portions of an organ or tissue)and cells isolated from a biological subject or from a particular region(e.g., a region containing diseased cells, or circulating tumor cells)of a biological subject. The biological sample that contains orsuspected to contain the target DNA may be of eukaryotic origin,prokaryotic origin, viral origin or bacteriophage origin. For example,the target DNA may be obtained from an insect, a protozoa, a bird, afish, a reptile, a mammal (e.g., rat, mouse, cow, dog, guinea pig, orrabbit), or a primate (e.g., chimpanzee or human). The target DNA mayalso be a cDNA (complementary DNA). The cDNA may be generated from anRNA template (e.g., mRNA, ribosomal RNA) using a reverse transcriptaseenzyme. The DNA product generated by another reaction, such as aligation reaction, a PCR reaction, or a synthetic DNA may also serve asthe target DNA. The target DNA may be dispersed in solution or may beimmobilized on a solid support, such as in blots, assays, arrays, glassslides, microtiter, on beads or ELISA plates.

As used herein, the term “contaminating nucleic acid” refers to anundesirable nucleic acid (e.g., a nucleic acid other than the targetDNA), which may interfere in a target DNA amplification reaction or maycompete with the target DNA in a DNA amplification reaction. Thecontaminating nucleic acid may be present in a reagent, a reagentsolution, or an apparatus that is used for target DNA amplification. Inother words, a contaminating nucleic acid is any nucleic acid, which isnot intended to be amplified, further characterized, or present in anassay to be performed. The contaminating nucleic acid may a RNA or DNA.For example, a DNA that is present in a reagent or reagent solution thatis used for performing a DNA synthesis reaction, prior to adding atarget DNA to be amplified, is considered to be a contaminating nucleicacid. The contaminating nucleic acid may also be a prior amplicon from aprevious amplification reaction. The contaminating nucleic acid in a DNAsynthesis reaction may act as a potential DNA template or may act as aprimer and thus may participate in the DNA synthesis reaction, resultingin unwanted amplification products. It is therefore desirable to removeany such contaminating nucleic acid prior to addition of the target DNAto the DNA amplification reaction mixture so that the contaminatingnucleic acid will not interfere with the DNA amplification reaction.Prior removal of contaminating DNA from the reagents and/or reagentsolutions is particularly desired to reduce artifacts during DNAsynthesis reaction if the DNA template to be amplified is available onlyin limited amounts.

As used herein, the term “DNA amplification reaction mixture” refers toa mixture of reagents that is essential for performing a DNAamplification reaction. The DNA amplification reaction mixture disclosedherein includes, at the minimum, at least one exonuclease-resistant,inosine-containing primer, at least one target DNA, dNTPs, at least oneendonuclease that is capable of nicking an inosine-containing strand ofa double stranded DNA at a reside 3′ to the inosine residue and at leastone DNA polymerase having strand displacement activity. It may furtherinclude reagents such as buffer(s), salt(s) and other components (e.g.,accessory proteins such as single stranded DNA binding protein,denaturant like urea, glycerol or pyrrolidine) that are required for atypical DNA amplification reaction.

As used herein, the term “decontaminating” refers to altering ormodifying a contaminating nucleic acid that may be present in a solutionor a reaction mixture such that it cannot interfere or compete with thetarget DNA in a subsequent DNA amplification reaction. Decontaminationmay also refer to rendering a contaminating nucleic acid inert. Thedecontamination can be affected by chemical modification of acontaminating nucleic acid, for example, by removing of one or morefunctional groups so that the contaminating nucleic acid is unable toreact with a DNA template (for it to act as a primer) or a DNApolymerase (for it to act as a DNA template). The contaminating nucleicacid may also be rendered inert by degrading or digesting the nucleicacid. Depending on the nature of the reagents employed, the mechanism bywhich the nucleic acid is rendered inert may vary. For example, acontaminating nucleic acid may be rendered inert by digesting thecontaminating nucleic acid with an exonuclease to produce freenucleotides having a 3′-hydroxyl group and a 5′-phosphate group. Thedecontamination may result either in a reduction in the amount of thecontaminating nucleic acid or a complete removal of the contaminatingnucleic acid. The decontamination process often leads to nucleotides ornucleotide fragments, which cannot act as a primer or serve as atemplate for a subsequent DNA amplification reaction.

As used herein, the term “primer” refers to a short linearoligonucleotide that hybridizes to a target nucleic acid sequence (e.g.a target DNA) to prime a nucleic acid synthesis reaction. The primer maybe an RNA oligonucleotide, a DNA oligonucleotide, or a chimericsequence. The primer may contain natural, synthetic, or modifiednucleotides. For example, the primer may comprise naturally occurringnucleotides (G, A, C or T nucleotides) or their analogues. Both theupper and lower limits of the length of the primer sequence areempirically determined. The lower limit on primer length is the minimumlength that is required to form a stable duplex upon hybridization withthe target nucleic acid under nucleic acid amplification reactionconditions. Very short primers (usually less than 3 nucleotides long) donot form thermodynamically stable duplexes with target nucleic acidsunder such hybridization conditions. The upper limit is often determinedby the possibility of having a duplex formation in a region other thanthe pre-determined nucleic acid sequence in the target nucleic acid.Generally, suitable primer lengths are in the range of about 4nucleotides long to about 40 nucleotides long. In some embodiments theprimer ranges in length from 5 nucleotides to 30 nucleotides. The term“forward primer” refers to a primer that anneals to a first strand ofthe target DNA and the term “reverse primer” refers to a primer thatanneals to a complimentary, second strand of the target DNA. Together, aforward primer and a reverse primer are generally oriented on the targetDNA sequence in a manner analogous to PCR primers, such that a DNApolymerase can initiate the DNA synthesis resulting in replication ofboth strands.

As used herein, the term “inosine-containing primer” refers to a primercomprising at least one inosine residue in its sequence. The inosineresidue is a 2′-deoxyribonucleoside or 2′-ribonucleoside residue whereinthe nucleobase is a hypoxanthine. The inosine residue is capable of basepairing with a thymine, an adenine, a cytidine or a uridine residue. Theinosine residue may also be an inosine analogue. For example, xanthinestructures are alternate structures to inosine residues, resulting fromdeamination of guanine. Inosine analog refers to a2′-deoxyribonucleoside or 2′-ribonucleoside wherein the nucleobaseincludes a modified base such as xanthine, uridine, oxanine (oxanosine),other O-1 purine analogs, N-6-hydroxylaminopurine, nebularine, 7-deazahypoxanthine, other 7-deazapurines, and 2-methyl purines. The inosine orinosine analogue residue may be positioned near the 3′ terminal end of aprimer sequence, often as a penultimate nucleotide at the 3′ end of theprimer sequence.

As used herein, the term “exonuclease resistant, inosine-containingprimer” refers to a primer sequence that contains at least one inosineresidue and is also resistant to the action of an exonuclease enzyme(i.e., not degraded by the exonuclease). In some embodiments, the primeris resistant to both 3′→5′ exonuclease activity and 5′→3′ exonucleaseactivity. In some embodiments, the primer is resistant to 3′→5′exonuclease activity. The inosine-containing primer may be engineered tomake it exonuclease-resistant by chemical modification, for example, byintroduction of at least one phosphorothioate linkage at an appropriateposition. For example, an inosine-containing primer that contains aphosphorothioate linkage between the 3′ terminal nucleotide and thepenultimate residue is resistant to the action of exonuclease in a 3′→5′direction. Since the 3′→5′ exonuclease digests the nucleic acid in a 3′to 5′ direction, the phosphorothioate linkage at this position preventsthe digestive action of the exonuclease. If the inosine residue ispresent on the 5′ side of the phosphorothioate, it will be maintained inthe primer. Similarly, an inosine-containing primer that contains aphosphorothioate linkage between the 5′ terminal nucleotide and thepenultimate residue is resistant to the action of exonuclease in a 5′→3′direction. When exonuclease-resistant, inosine containing primerhybridizes with a target DNA and forms double stranded nucleic acidstructure, the double stranded structure may be recognized by specificendonucleases resulting in a single stranded nick in theinosine-containing strand. For example, endonuclease V is capable ofnicking the inosine-containing strand of a double stranded DNA at aposition 3′ to the inosine residue when the exonuclease-resistantinosine-containing primer is hybridized to a target DNA.

As used herein, the term “dNTPs” refers to a mixture of deoxynucleotidetriphosphates that act as precursors required by a DNA polymerase forDNA synthesis. Each of the deoxynucleotide triphosphates in a dNTPmixture comprises a deoxyribose sugar, an organic base, and a phosphatein a triphosphate form. A dNTP mixture may include each of the naturallyoccurring deoxynucleotide triphosphate (e.g., dATP, dTTP, dGTP, dCTP ordUTP). In some embodiments, each of the naturally occurringdeoxynucleotide triphosphates may be replaced or supplemented with asynthetic analog; provided however that inosine base may not replace orsupplement guanosine base (G) in a dNTP mixture. Each of thedeoxynucleotide triphosphates in dNTP may be present in the reactionmixture at a final concentration of 10 μM to 20,000 μM, 100 μM to 1000μM, or 200 μM to 300

As used herein, the term “amplicon” refers to nucleic acid amplificationproducts that result from the amplification of a target nucleic acid.Amplicons may comprise a mixture of amplification products (e.g. a mixedamplicon population), several dominant species of amplification products(e.g. multiple, discrete amplicons), or a single dominant species ofamplification product. A single species of amplicon may be isolated froma mixed population of amplicons using art-recognized techniques, such asaffinity purification or electrophoresis. An amplicon may comprisesingle-stranded or double-stranded DNA, DNA:RNA hybrids or RNA dependingon the reaction scheme used. An amplicon may be largely single-strandedor partially double-stranded or completely double-stranded DNA, DNA:RNAhybrids, or RNA.

As used herein, the term “terminal nucleotide” refers to a nucleotidethat is located at a terminal position of an oligonucleotide or primersequence. The terminal nucleotide that is located at a 3′ terminalposition of an oligonucleotide sequence is referred as a 3′ terminalnucleotide, and the terminal nucleotide that is located at a 5′ terminalposition is referred as a 5′ terminal nucleotide. The nucleotide that islocated at a penultimate position refers to a nucleotide that isimmediately adjacent to a terminal nucleotide. For example, NNNNNNIAdepicts a nucleotide sequence (an octamer) in which the inosine residue(I) is at a penultimate 3′ position and an adenosine residue is the 3′terminal nucleotide.

The term “mutant endonuclease” or “engineered endonuclease” as usedherein refers to an endonuclease enzyme that is generated by geneticengineering or protein engineering, wherein one or more amino acidresidues are altered from the wild type endonuclease. The alteration mayinclude a substitution, a deletion or an insertion of one or more aminoacid residues. Throughout the specification and claims, the substitutionof an amino acid at one particular location in the protein sequence isreferred using a notation “(amino acid residue in wild type enzyme)(location of the amino acid in wild type enzyme) (amino acid residue inengineered enzyme)”. For example, a notation Y75A refers to asubstitution of a Tyrosine (Y) residue at the 75^(th) position of thewild type enzyme by an Alanine (A) residue (in mutant/engineeredenzyme).

The term “conservative variants”, as used herein, applies to both aminoacid and nucleic acid sequences. With respect to particular nucleic acidsequences, the term “conservative variants” refers to those nucleicacids that encode identical or similar amino acid sequences (i.e., aminoacid sequences that have similar physico-chemical properties) andinclude degenerate sequences. For example, the codons GCA, GCC, GCG, andGCU all encode alanine. Thus, at every amino acid position where analanine is specified, any of these codons may be used interchangeably inconstructing a corresponding nucleotide sequence. Such nucleic acidvariants are conservative variants, since they encode the same protein(assuming that is the only alternation in the sequence). One skilled inthe art recognizes that each codon in a nucleic acid, except for AUG(sole codon for methionine) and UGG (tryptophan) may be modifiedconservatively to yield a functionally identical peptide or proteinmolecule. As to amino acid sequences, one skilled in the art willrecognize that alteration of a polypeptide or protein sequence viasubstitutions, deletions, or additions of a single amino acid or a smallnumber (typically less than about ten) of amino acids may be a“conservative variant” if the physico-chemical properties of the alteredpolypeptide or protein sequence is similar to the original. In somecases, the alteration may be a substitution of one amino acid with achemically similar amino acid. Examples of conservative variantsinclude, but not limited to, the substitution of one hydrophobic residue(e.g., isoleucine, valine, leucine or methionine) for one another; orthe substitution of one polar residue for another (e.g., thesubstitution of arginine for lysine, glutamic for aspartic acid, orglutamine for asparagine) and the like. Genetically encoded amino acidsgenerally may be divided into four families: (1) acidic: aspartate,glutamate; (2) basic: lysine, arginine, histidine; (3) nonpolar:alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan; and (4) uncharged polar: glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine.

One or more embodiments of the methods and kits forendonuclease-assisted DNA amplification assays comprises use of anendonuclease that is capable of nicking an inosine-containing strand ofa double-stranded nucleic acid at a location 3′ to the inosine residue.In some embodiments, the endonuclease is a genetically engineeredendonuclease. The DNA amplification assays described herein furtheremploy an exonuclease-resistant primer that comprises at least oneinosine residue. The exonuclease-resistant primer solution may bedecontaminated prior to the DNA amplification reaction by treating itwith an appropriate exonuclease.

For DNA amplification assays, samples suspected or known to contain aparticular target DNA may be obtained from a variety of sources. Thesample may be, for example, a biological sample, a food, an agriculturalsample, or an environmental sample. Samples may also be derived from avariety of biological subjects, including prokaryotic or eukaryoticorigin and includes viruses. The sample may be derived from a biologicaltissue or a body fluid or an exudate (e.g., blood, plasma, serum orurine, milk, cerebrospinal fluid, pleural fluid, lymph, tears, sputum,saliva, stool, lung aspirates, throat or genital swabs, and the like),whole cells, circulating tumor cells, cell fractions, or cultures.

The target DNA for a nucleic acid assay may be dispersed in solution orimmobilized on a solid support (such as blots, paper punches, arrays,microtiter, or well plates). The target DNA may either besingle-stranded or double-stranded. The target DNA template can be acircular DNA, a linear DNA or a nicked DNA. The DNA template may be agenomic DNA, a plasmid DNA or a cDNA. The target DNA may be pretreatedto make it available for hybridization with the primer. For example,when a target DNA is in a double stranded form, it may be denatured togenerate a single stranded form of the target DNA. The target doublestranded DNA may be thermally or chemically denatured, or both thermallyand chemically denatured. In some embodiments, the double stranded DNAis chemically denatured using a denaturant (e.g., glycerol, ethyleneglycol, formamide, or a combination thereof) that reduces the meltingtemperature of double stranded DNA. In certain embodiments, thedenaturant reduces the melting temperature by 3° C. to 6° C. for every10% (vol./vol.) of the denaturant added to the reaction mixture. Thedenaturant or combination of denaturants may comprise 1%, 5%, 10%(vol./vol.), 15% (vol./vol.), 20% (vol./vol.), or 25% (vol./vol.) ofreaction mixture. In certain embodiments, the denaturant comprisesethylene glycol. In alternative embodiments, the denaturant is acombination of glycerol (e.g., 10%) and ethylene glycol (e.g., 15% to20%). Salts that reduce hybridization stringency may be included in thereaction buffers at low concentrations to chemically denature the targetDNA is at low temperatures. In embodiments where the target DNA isthermally denatured the denaturing step comprises thermally denaturingthe target DNA (e.g., by heating the target DNA at 95° C.).

In some embodiments, an endonuclease-assisted DNA amplification methodproduces at least one amplicon based on a target DNA. The target DNA isfirst hybridized with an exonuclease-resistant, inosine-containingprimer followed by amplification of the target DNA using a DNApolymerase (e.g., a strand displacement polymerase) and dNTPs inpresence of an endonuclease that is capable of nicking aninosine-containing strand of a double-stranded nucleic acid at alocation 3′ to the inosine residue. The dNTPs provide a combination ofdeoxyribonucleotides required by the DNA polymerase for DNA synthesis.DNA polymerases use dNTP mixture to add nucleotides to the 3′ hydroxylend of a primer annealed to a template strand of DNA in a complementaryfashion, creating a new DNA strand (amplicon) complementary to thetarget DNA template. In some embodiments, each of the naturallyoccurring deoxynucleotides may be replaced or supplemented with asynthetic analog; provided however that deoxyinosinetriphosphate (dITP)may not replace or supplement dGTP in the dNTP mixture. The product ofDNA amplification reaction may be single stranded or double-strandedDNA, often extending to the end of the template strand. The inosinenucleotide in the inosine-containing primer may be positioned at least 4nucleotides, at least 5 nucleotides, or at least 10 nucleotidesdownstream of the 5′ end of the inosine-containing primer. For example,the inosine containing random octamer primer may have a sequence such asNNNINNNN or NNNNINN. In certain embodiments, the inosine nucleotide maybe the penultimate 3′ nucleotide of the primer. In alternativeembodiments, inosine may be present at both the penultimate 3′ residueand ultimate 3′ residue. In some embodiments, the inosine-containingprimer comprises an inosine analogue. In some embodiments, the DNAamplification method comprises the steps of providing a target DNA and aprimer solution comprising at least one exonuclease-resistant,inosine-containing primer; generating a DNA amplification reactionmixture by mixing together the target DNA, the primer solution, at leastone 5′→3′ exonuclease-deficient DNA polymerase having stranddisplacement activity, and at least one endonuclease that is capable ofnicking an inosine-containing strand of a double stranded DNA at aresidue 3′ to an inosine residue; and amplifying at least one portion ofthe target DNA using the DNA amplification reaction to produce at leastone amplicon. The at least one exonuclease-resistant, inosine-containingprimer may be a forward primer or a reverse primer or a mixture of both.Apart from the exonuclease-resistant, inosine-containing primer, theprimer solution may include other essential nucleic acid amplificationreagents such as dNTPs and buffers. The primer solution may also includeone or more of reagents that enhance or assist DNA amplificationreaction such as single strand DNA binding protein (SSB protein),formamide, ethylene glycol, or Ficoll. Before generating the DNAamplification mixture, the primer solution, may be decontaminated bytreating the primer solution with an appropriate exonuclease to removeany contaminating nucleic acid. Further, after the decontaminationreaction, the added exonuclease may be inactivated prior to the DNAamplification reaction. In some embodiments, the target DNA is amplifiedby employing an isothermal amplification method.

FIG. 1 depicts a schematic representation of an embodiment ofendonuclease-assisted target DNA amplification. Upon binding of aninosine-containing primer to the target DNA, the DNA polymerase (e.g., a5′→3 exonuclease-deficient Bst DNA polymerase) extends theinosine-containing primer thereby generating a double stranded DNA(primer extension product). The extension reaction creates a nickingsite for an endonuclease, which is capable of creating a single strandednick at the inosine-containing strand of a double stranded DNA at aposition 3′ to the inosine residue. The endonuclease nicks the doublestranded DNA at this nicking site. Nicking creates a new DNA synthesisinitiation site for the DNA polymerase. The DNA polymerase binds to thisinitiation site and further elongates the nicked primer. Since the DNApolymerase has strand displacement activity, it displaces asingle-stranded DNA product while it re-creates the double-strandedprimer extension product. This cycle repeats, synthesizing multiplesingle strands of DNA complementary to the downstream portion of thetarget DNA template. The schematic representation of a nucleic acidamplification shown in FIG. 1 may be varied by employing additionalprimers or other oligonucleotides, additional enzymes, additionalnucleotides, stains, dyes, or other labeled components. For example,amplification with a single primer may be used for dideoxy sequencing,producing multiple sequencing products for each molecule of template,and, optionally by the addition of dye-labeled dideoxynucleotideterminators. Labeled probes may be generated from double-stranded cDNAmade with a sequence-tagged oligo dT primer from mRNA samples. A singleprimer may be the complement of the tag sequence, facilitatingidentification and/or isolation. An endonuclease V may be used as asuitable endonuclease for this reaction. Endonuclease V is a repairenzyme that recognizes DNA containing inosines (or inosine analogues)and hydrolyzes the second or third phosphodiester bonds 3′ to theinosine (i.e., specifically nicks a DNA at a position two nucleotides 3′to an inosine nucleotide, about 95% the second phosphodiester bond andabout 5% the third phosphodiester bond) leaving a nick with 3′-hydroxyland 5′-phosphate. When the target DNA is double stranded the nick occursin the strand comprising the inosine residue.

In some embodiments, a strand displacement DNA polymerase, anendonuclease V and an exonuclease-resistant, inosine-containing primerare employed in the DNA amplification reaction. For DNA amplification,the exonuclease-resistant, inosine-containing primer hybridizes with thetarget DNA. Inosine residue in the primer may base pair with a cytidineresidue or a thymidine residue in the target DNA, wherein hypoxanthenesubstitutes for a guanine to complement a cytosine; or substitutes foran adenine to complement a thymine. A complimentary strand to the targetDNA template is then generated by DNA synthesis thereby generating adouble-stranded DNA. Generation of the double stranded DNA in turngenerates a nicking site for the endonuclease V. The endonuclease Vnicks the inosine-containing strand of this double-stranded DNA. The DNApolymerase then once again generates the complementary strand from thenicked position. This elongation step once again creates a nicking sitefor the endonuclease V. Thus the elongation by the DNA polymerasefollowed by nicking by the endonuclease V gets repeated multiple timesuntil any one of the essential DNA amplification reagents in the DNAamplification reaction mixture is exhausted. In each cycle, the stranddisplacement DNA polymerase employed in these reactions displaces thecomplementary strand that was generated in the previous cycle. The stepsof hybridization, elongation, nicking and further elongation may occursubstantially simultaneously. Thus, one or more embodiments of themethods comprise an inosine residue that is introduced into a specificposition of a target nucleic acid (via an oligonucleotide primer),followed by repeated generation of complimentary strand of the targetnucleic acid using a polymerase and an endonuclease V that nicks thegenerated double stranded nucleic acid at the inosine-containing strandto initiate a second cycle of complementary strand generation by thepolymerase.

FIG. 2 depicts a schematic representation of an embodiment of theendonuclease-assisted amplification reaction usingexonuclease-resistant, inosine-containing primer. The sequence of theexonuclease-resistant, inosine-containing primers used in theamplification reaction typically depends on the sequence of the DNAtemplate to be amplified and/or the desired type of amplification (e.g.,random vs. specific). In some embodiments, the exonuclease-resistant,inosine-containing primer used herein comprises at least one inosineresidue located at least 4 nucleotides downstream of the 5′ terminalresidue. The exonuclease-resistant primers may include modifiednucleotides, which make them resistant to the exonuclease digestion. Insome embodiments, the primer contains at least one nucleotide that makesthe primer resistant to degradation by an exonuclease, particularly by a3′5′ exonuclease. The modified nucleotide may be a phosphorothioatenucleotide. For example, an exonuclease resistant primer may possessone, two, three or four phosphorothioate linkages between nucleotides inthe sequence (e.g., NNNNN*N*N*I*N or N*NNNN*N*N*I*N). The modifiednucleotide is commonly a 3′-terminal nucleotide of the primer sequencehaving a penultimate inosine residue (e.g., (NNN)_(n)NI*N or(NNN)_(n)NI*I where * represents a phosphorothioate bond between thenucleotides and the integer value of n may range depending on the lengthof the primer used, for example, the value of n may range from 0 to 13).However in some embodiments the primer could have the modifiednucleotide as the inosine residue (e.g., NNNNNN*IN). In someembodiments, the modified nucleotide may be located at a position otherthan the 3′-terminal position provided that the primer sequence containsat least one inosine residue located next to the modified residue (e.g.,NNNNI*NNNN or NNNN*INNNN). When the modified nucleotide is located atpositions other than the 3′-terminal end of a primer sequence, the3′-terminal nucleotide of said primer may be removed by the 3′5′exonuclease activity. Some endonuclease V may have an associated 3′→5′exonuclease activity. The thioated inosine primers may prevent theendonuclease V from removing the inosine. Other nucleotide modificationsknown in the art that make a nucleotide sequence resistant to anexonuclease may be used as well.

Suitable inosine-containing primers that are also exonuclease-resistantmay be designed and selected depending on the sequence and the nature ofthe DNA template to be amplified. Exonuclease-resistant,inosine-containing primers may be synthesized using any of theart-recognized synthesis techniques. Amplicons may be generated using asingle inosine-containing primer, paired inosine-containing primers, ornested-paired inosine-containing primers that are exonuclease resistant.Primer design software such as AutoDimer™ may be employed to design asingle primer or multiple primers that are capable of annealing to anucleic acid and facilitating polymerase extension. Theexonuclease-resistant, inosine-containing primer may be designed in sucha way that the melting temperature of the primer is about 50° C. with asalt concentration of about 6 mM. In some embodiments, relatively shortprimers (e.g., 10-mers to 20-mers; more preferably 14-mers to 18-mers,most preferably 16-mers) may be employed. The exonuclease-resistant,inosine-containing primer may either be a specific primer or a randomprimer. For whole genome amplification reaction, a random primer mixturecomprising primers generated by randomizing all the residues other thanthe inosine residue may be used. Either a single primer or multipleprimers may be employed for amplification. Specific primers have, or areengineered to have, a nucleotide sequence that is complementary, in theWatson-Crick sense, to a pre-determined sequence, which is present inthe target DNA template.

In some embodiments, the exonuclease-resistant, inosine-containingprimer is designed such that the inosine residue is positioned in theprimer at a location complementary to a cytosine residue in the targetDNA. In some embodiments, the inosine appears as the penultimate 3′ baseof the primer. Because the reaction conditions (e.g., temperature andionic strength) affect annealing of primer to target DNA, optimalpositioning of the inosine in the primer may be adjusted according tothe reaction conditions. In general, the inosine residue is positionedaway from the 5′ end of the prime such that the primer remains annealedto the target DNA after nicking by the endonuclease (e.g., the length ofthe nicked primer is sufficient to enable binding to the target DNAunder the nucleic acid assay reaction conditions). Accordingly, thesegment of the primer 5′ of the inosine should have a meltingtemperature approximately equal to the reaction temperature at thechosen reaction conditions. In some embodiment, the inosine-containingprimer may comprise more than one inosine residue or inosine analogues.If there are two template Gs in a row, two inosines may appear in theprimer as the both the penultimate 3′ and the final residues. In thiscase, nicking by the endonuclease 2 nucleotides 3′ to either inosineresidues would have the same effect of creating a nicked DNA strand. Theinosine residues may be located both at the penultimate 3′ position andthe terminal 3 position. These two inosines may be linked byphosphorothioate linkages. In some embodiments, the inosine-containingprimer may demonstrate a melting temperature of 25° C. to 80° C., 30° C.to 65° C., or 40° C. to 55° C. in the reaction mixture. In someembodiments, the exonuclease-resistant, inosine-containing primerdemonstrates a melting temperature of 50° C. in the reaction mixture.

With a single, forward primer, the rate of synthesis of complimentarycopies of target DNA is relatively constant, resulting in a steady,linear increase in the number of complimentary copies with time.Multiple primers, each of them containing at least one inosine residueand are exonuclease-resistant, may be included in the reaction mixturein some embodiments to accelerate the amplification process. Embodimentswhere both the plus and minus strands are generated, paired primerscomprising a forward primer and a reverse primer may be included in thereaction mixture. For example, when a reverse primer (a primer thatanneals to the generated complementary strand ((+) strand) to furthergenerate a (−) strand in the reverse direction) that anneals to thecomplementary strand of target DNA at a defined distance from theforward primer is added, the amplification process is accelerated. Sincethe targets for each of these primers would be present in the originaltemplate, both strands would be amplified in the two primer scheme(“Ping product” being the amplicon of the forward primer and the “Pongproduct” being the amplicon of the reverse primer). The inclusion ofmultiple paired primers may improve the relative percentage of adiscrete product in the reaction mixture. The internal most forward andinternal most reverse primers may be placed relatively close to eachother (e.g., less than about 50 bases apart), minimizing the timerequired to complete the forward amplicon ((+) strand) to its 5′ end asdefined by the endonuclease V cleavage site, and thereby reducing thetotal time required to generate amplicons from the target DNA. Thereaction rate reaches a maximum when the amount of nuclease, polymerase,or any other component(s) becomes limiting. Additional pairs of nestedprimers, each of them exonuclease-resistant and containing at least oneinosine residue, may also be used to further increase amplificationrates or increase the specificity of the reaction. Nested primers may bedesigned to bind at or near the 3′ end of the previous amplicon so thatin a series, each primer in the series will hybridize next to each otheron the original target. Where multiple nested primers are used, SSBprotein at a concentration of 1 ng to 10 μg in a 10 μL volume may beincluded in the reaction mixture to increase fidelity and to reducebackground. Amplification with multiple, paired primers facilitatesrapid and extensive amplification, which is useful to detect thepresence of specific sequences, to quantify the amounts of thosesequences present in a sample, or to produce quantities of a sequencefor analysis by methods such as electrophoresis for size measurement,restriction enzyme digestion, sequencing, hybridization, or othermolecular biological techniques.

In some embodiments, extender templates, which are specific primersequences (e.g., primers that contain additional 5′ sequences that allowfor additional sequence to be added to the end of hybridized ampliconDNA, and are used to generate a promoter sequence or a restrictionendonuclease site specific sequence or a novel primer binding sitesequence), may be annealed at the 3′ end of the amplicon byincorporating in an exonuclease-resistant, inosine-containing primer. Anextender template may be designed such that it anneals to the 3′ end ofan amplicon. If the extender template contains two stretches ofsequences, one complementary to the amplicon, and one that is not,hybridization will create a 5′ overhang of the non-complementary primersequence. The 3′ recessed end of the amplicon can then be furtherextended by the DNA polymerase. This extension reaction may be employedto incorporate specific DNA sequences at the 3′ end of an amplicon. Insome embodiments, the 5′ end of the extender template may contain ahairpin loop, with a fluorescent dye and a quencher located on eitherarm of the stem such that the dye fluorescence is largely quenched byresonance energy transfer. Upon extension of the recessed 3′ end of theamplicon by a DNA polymerase, the stem-loop structure gets converted toa double stranded structure and causes the dye and the quencher to beseparated further. This eliminates some or all of the fluorescencequenching, and thus generates a detectable signal. This signal may bemultiplexed by appropriate sequence selection of the extender templatesand the color of the quenched dyes so that 2 or more independentamplification processes may be monitored simultaneously. In someembodiments the 5′ end of the extender template may include thecomplement of an RNA polymerase promoter sequence. Thus, a doublestranded RNA polymerase promoter may be generated by hybridizing theextender template to the amplicon followed by extension of the recessed3′ end of the amplicon by the DNA polymerase. If an RNA polymerase isincluded in the reaction, the amplicon may be then transcribed as asingle-stranded RNA polymerase template to generate corresponding RNAs.

Since the exonuclease-resistant, inosine-containing primer is resistantto the action of exonuclease, the solution containing the primer can bepre-treated with an exonuclease before adding to the DNA amplificationreaction mixture. The exonuclease treatment removes or reduces anycontaminating nucleic acid that may be present in the primer solution.The solution containing the exonuclease-resistant, inosine-containingprimer may further comprise other reagents required for a DNAamplification reaction described herein (except the target DNA) such asdNTPs, DNA polymerase, endonuclease, buffers and/or singlestrand-binding proteins. If DNA polymerase is included in the primersolution, it should be selected such that it can withstand theexonuclease inactivation step. Further, if the a proofreading DNApolymerase such as Phi29 DNA polymerase is used as an exonuclease in thedecontamination step, primer solution should not contain any dNTPs. Thedecontamination of the primer solution if often achieved by incubatingthe primer solution with an exonuclease and a divalent cation to allowthe exonuclease to render the contaminating nucleic acid inert. A singleexonuclease or a combination of exonucleases may be used todecontaminate the primer solution. In some embodiments, one or more ofexonuclease may be used in the reaction. Suitable exonucleases that maybe used in the present invention include, but not limited, to Phi29 DNApolymerase, exonuclease I, exonuclease III, exonuclease VII, T7 gene-6exonuclease, spleen exonuclease, T5 D15 exonuclease and lambdaexonuclease. In one embodiment, a combination of exonuclease I andexonuclease III is used in the decontaminating the primer solution. WhenPhi29DNA polymerase is used as the suitable exonuclease, it may beinactivated very easily by incubating the primer solution at 45 degrees,so the Phi29 DNA polymerase is inactive at the temperature used for DNAamplification.

Any divalent cation that can activate the exonuclease can be used in thedecontamination reaction. Some non-limiting examples include magnesiumand manganese. The concentration of the divalent cation primarilydepends on the concentration of the exonuclease. Some of the parametersthat determine the concentration of the exonuclease include the amountof contaminating nucleic acid, the turn-over of the particularexonuclease and other kinetic parameters for the exonuclease activity.In some embodiments, a molar excess of the divalent cation with respectto the exnuclease is used for decontaminating the primer solution. Theprimer solution containing the exonuclease-resistant, inosine-containingprimer is incubated with the exonuclease and the divalent cation for aperiod of time that is sufficient to render the contaminating nucleicacid inert. The incubation time may vary with the kinetic properties ofthe exonuclease and the divalent cation that is being used. Theincubation time may also depend on the temperature at which theincubation is performed. Incubation time may be optimized by analyzingthe efficiency of the de-contamination process by employing any of thetechniques known in the art for characterizing the presence of nucleicacids. Suitable incubation times may range from about 5 min to about 3h. In some embodiments, the incubation time may range from about 1 minto about 100 min. In some specific embodiments, the primer solutioncontaining the exonuclease-resistant, inosine-containing primer may beincubated with the exonuclease and the divalent cation at 37° C. forabout 60 min. The temperature at which the incubation of the primersolution is performed may vary by the nature of the particularexonuclease used. The maximum temperature that may be used for theincubation is limited by the stability of the exonuclease and theminimum temperature that may be employed for the incubation is decidedby the exonuclease activity at that temperature. In some embodiments,the incubation is performed at a temperature at or below 50° C. In someembodiments, the suitable incubation temperature ranges from about 0° C.to about 45° C. In some specific embodiments, the incubation may beperformed at a temperature between about 10° C. to about 40° C.

In some embodiment, a suitable endonuclease may be added to the primersolution along with the exonuclease for decontamination. This isparticularly useful in embodiments wherein the contaminating nucleicacid may include a circular DNA or wherein the entire backbone of theprimer is nuclease resistant. Endonucleases act on circular DNA and nickthem. Once the nick is made, the exonuclease can act on the contaminant,nicked DNA and degrade them to make them inert. Non limiting examples ofsuitable endonucleases include DNAses such as DNAse I.

In some embodiments, a SSB protein may be added along with theexonuclease to the primer solution containing exonuclease-resistant,inosine containing primer. Suitable SSB proteins that may include, butnot limited to, extreme thermostable single stranded DNA-binding protein(ET SSB from New England Biolabs, MA), rec A (e.g., E. coli RecA), TthRecA (RecA homolog isolated from Thermus thermophilus from New EnglandBiolabs, MA), phage T4 gene-32 protein, T7 gene 2.5 protein, Ncp7, andE. coli SSB protein. The addition of exonuclease, divalent cation and/orthe SSB to the solution comprising exonuclease-resistant primer mayeither be performed sequentially or simultaneously. In embodiments wherethe sequential addition is performed, the addition may be carried out inany particular order. For example, in some embodiments, the exonucleaseand the divalent cation may be mixed first and then added to the primersolution followed by the SSB protein. In some other embodiments, theprimer solution may be contacted with the SSB protein first and then theexonuclease and the divalent cation could be added.

After the decontamination step using the exonuclease, the exonuclease inthe primer solution may be inactivated. The exonuclease may beinactivated by a variety of methods available in the art. In onespecific embodiment, the exonuclease may be inactivated by thermaldenaturation of the exonuclease. This may be achieved by incubating thedecontaminated primer solution at a temperature at which the nuclease isnot stable. The incubation is performed for a specified period of timethat is sufficient to inactivate the exonuclease. In some embodiments,this may be achieved by incubating the decontaminated primer solution attemperatures at or above 65° C. In some embodiments, the decontaminatedprimer solution may be incubated at a temperature between 65° C. andabout 95° C. The time that is sufficient to thermally inactivate theexonuclease may vary depending on the temperature used and the type ofnuclease involved. Typically, the thermal inactivation may be performedfor a time span of about 30 sec. to about 2 h. In some embodiments, thedecontaminated primer solution may be incubated at about 85° C. for 15min and then at about 95° C. for 5 min. The time span and thetemperature may be optimized as required to thermally inactivate theexonuclease. In specific embodiments wherein the primer solutioncontains a DNA polymerase and/or endonuclease V along with otherreagents in the exonuclease treatment step for removing contaminatingnucleic acids, the DNA amplification reaction may be performed with orwithout inactivating the exonuclease. In embodiments wherein theinactivation step is not used, the quantity of exonuclease may beselected such that it does not interfere with the DNA amplificationreaction. In embodiments wherein the inactivation step needs to beperformed, the DNA polymerase, endonuclease V and exonuclease are eitherselected such that the exonuclease can selectively be inactivatedwithout inactivating the DNA polymerase and/or endonuclease or the DNApolymerase and endonuclease V is added to the decontaminated primersolution only after deactivating the exonuclease that was used fordecontamination.

Once substantially all the contaminating nucleic acid has been renderedinert, a DNA template to be amplified may be added to the decontaminatedprimer solution. At least one DNA polymerase having strand displacementactivity, at least one endonuclease that is capable of nicking aninosine-containing strand of a double stranded DNA at a residue 3′ tothe inosine residue and free nucleotides are also added to the primersolution if they are not already present in the primer solution duringthe decontamination step. Removal of degraded contaminating nucleicacids from the decontaminated primer solution may not be required sincethey do not interfere with the DNA synthesis reaction. The DNApolymerase that could be employed for amplifying the DNA template may bea proofreading DNA polymerase or a non-proofreading DNA polymerase. Insome specific embodiments, a combination of a proofreading DNApolymerase and a non-proofreading DNA polymerase may be used forefficient amplification of the DNA template.

In some embodiments, the DNA amplification reaction is performed underisothermal conditions. The reaction temperature during an isothermalamplification reaction condition may range 1° C., 5° C., or 10° C. froma set temperature. In some embodiments, the reaction temperature of DNAamplification is held at 45° C. (±1° C.). Thermally stable endonucleasesand thermally DNA polymerases may be used depending upon the reactiontemperature of DNA amplification reaction.

Any of the DNA polymerases known in the art may be employed for DNAamplification. DNA polymerases suitable for use in the inventive methodsmay demonstrate one or more of the following characteristics: stranddisplacement activity; the ability to initiate strand displacement froma nick; and/or low degradation activity for single stranded DNA. In someembodiments, the DNA polymerase employed may be devoid of one or moreexonuclease activities. For example, the DNA polymerase may be a 3′→5′exonuclease-deficient DNA polymerase or the DNA polymerase may lack5′→3′ exonuclease activity. In some embodiments, the DNA polymerase maylack both 3′→5′ and 5′→3′ exonuclease activities (i.e., an exo (−) DNApolymerase). Exemplary DNA polymerases useful for the methods include,without limitation, Klenow, 5′→3′ exonuclease-deficient Bst DNApolymerase (the large fragment of Bst DNA polymerase), 5′→3′exonuclease-deficient delta Tts DNA polymerase, exo (−) Klenow, orexo(−) T7 DNA polymerase (Sequenase™).

In some embodiments, a proofreading DNA polymerase may be used for DNAamplification reaction. In some specific embodiments, the solutioncontaining proofreading DNA polymerase may also be decontaminated priorto its addition to the decontaminated primer solution. Thedecontamination of the proof-reading DNA polymerase solution may beachieved by pre-treating it with a divalent cation (e.g., Mg2+ or Mn2+)in the absence of dNTPs. In one embodiment, a Phi29 DNA polymerase isemployed and Phi29 DNA polymerase solution is decontaminated byincubating the Phi29 DNA polymerase solution with magnesium ions at aspecified temperature for a sufficient period of time to render anycontaminating nucleic acid inert prior to its addition to thedecontaminated primer solution for carrying out the template DNAamplification.

Polymerase enzymes typically require divalent cations (e.g., Mg⁺², Mn⁺²,or combinations thereof) for nucleic acid synthesis. Accordingly, one ormore divalent cations may be added to the DNA amplification reactionmixture. For example, MgCl₂ may be added to the reaction mixture at aconcentration range of 2 mM to 6 mM. Higher concentrations of MgCl₂ maybe preferred when high concentrations (e.g., greater than 10 pmoles,greater than 20 pmoles, or greater than 30 pmoles) of inosine-containingprimer are included in the reaction mixture.

In some embodiments, a mutant endonuclease V is included in the DNAamplification reaction mixture to nick the inosine-containing doublestranded DNA. The mutant endonuclease V may be generated by anytechnique for genetic engineering or protein engineering includingsite-directed mutagenesis or artificial gene synthesis. The geneticengineering may include an alteration of one or more amino acid residuesof a wild type endonuclease V. The alteration may include substitution,insertion and/or deletion of one or more amino acid residues of the wildtype endonuclease V. Mutant endonuclease V may be generated by rationaldesign of protein or by directed evolution. In some embodiments, arationally designed, mutant endonuclease V enzyme is employed that hasincreased substrate binding, increased nicking efficiency, increasednicking specificity and/or increased nicking sensitivity. A mutantendonuclease V may also be designed such that the substrate binding isreversible. The mutant endonuclease V enzyme may then support repeatednicking by each enzyme, whereas the corresponding wild type enzyme maybe capable of only a single round (or a few limited rounds) of nicking(for example, the wild type E. coli endonuclease V (SEQ ID NO: 1)remains bound to the DNA after nicking). Such mutant endonuclease V maybe used in a reaction mixture in less than stoichiometric quantities toeffect a nicking reaction. In some embodiments, conservative variants ofthe mutant endonuclease V may be used for the DNA amplificationreaction. For example, further alteration of a mutant endonuclease V viasubstitution, deletion, and/or addition of a single amino acid or asmall number (typically less than about ten) of amino acids may be a“conservative variant” if the physico-chemical properties of the alteredmutant endonuclease V is similar to the original mutant endonuclease V.In some cases, the alteration may be a substitution of one amino acidwith a chemically similar amino acid.

In some embodiments, a mutant endonuclease V that preferentially nicksthe inosine-containing strand of a double stranded DNA at a position 3′to the inosine residue when the inosine residue is paired with acytosine residue may be used. In some other embodiments, a mutantendonuclease V that preferentially nicks the inosine-containing strandof a double stranded DNA at a position 3′ to the inosine residue whenthe inosine residue is paired with a thymine residue may be used. Themutant endonuclease V may have a higher efficiency than the wild typeendonuclease V to nick the inosine-containing strand of the doublestranded DNA when the inosine is paired with cytosine or thymine.Further, a mutant endonuclease V employed in the DNA amplificationreaction may preferentially nick an inosine-containing strand of adouble stranded DNA than an inosine-containing single stranded DNA. Forexample, Y75A E. coli mutant endonuclease V (SEQ ID NO: 2) nicks adouble stranded DNA comprising an inosine residue better than a singlestranded DNA comprising an inosine residue. In contrast, Y80A Tma mutantendonuclease V (SEQ ID NO: 6) nicks a single stranded DNA comprising aninosine residue better than a double stranded DNA comprising an inosineresidue. Some mutant endonucleases may nick structures other than DNAsequences containing inosine residue while some others may be veryspecific to inosine-containing DNA sequences. For example, Tma and Afuendonucleases (SEQ ID NO: 3 and SEQ ID NO: 5) do not nick structuressuch as flaps and pseudo Y structures. In some embodiments, when thereare multiple inosine residues in a double stranded DNA, the employedendonuclease V mutant may preferentially nick (often 1 or 2 nucleotides3′ to the inosine residue) the inosine residue that is paired with acytosine residue than the inosine residue that is paired with a thymineresidue. In some aspects, the endonuclease V mutant may nick a doublestranded DNA containing base pair mismatches. The nicking may happen atthe location of the base pair mismatch or at a location 3′ to the basepair mismatch that is separated by one or more bases.

In some embodiments, a heat stable endonuclease V is used for the DNAamplification reaction. For example, in a DNA amplification assay, wherethermal denaturation (either partial or full denaturation) of a targetDNA is performed, a heat stable endonuclease V or a heat stableendonuclease V mutant may be preferred. In other embodiments wherethermal denaturation of a target DNA is not required, a wild typeendonuclease V or an endonuclease V mutant (e.g., Y75A mutant E. coliendonuclease V) that has maximum enzymatic activity at a relatively lowtemperature (e.g., 45° C.) may be used. For example, Y75A E. Coliendonuclease V mutant is inactivated by incubation at 50° C., whereas itretains its enzymatic activity at 37-45° C. Afu endonuclease V (bothwild type (SEQ ID NO: 3) and Y75A mutant (SEQ ID NO: 4)) or Tmaendonuclease V (both wild type (SEQ ID NO: 5) and Y80A mutant (SEQ IDNO: 6)) are generally more thermo stable than the E. coli endonuclease V(both wild type (SEQ ID NO: 1) and Y75A mutant (SEQ ID NO: 2)). In someembodiments where strand displacement DNA synthesis by DNA polymerasemay be increased by incubation at an elevated temperature, anendonuclease V which functions at high temperature (e.g., 45-80° C.) maybe used.

In some embodiments, a mutant E. coli endonuclease V is employed for DNAamplification reactions. The mutant E. coli endonuclease may be a Y75Amutant E. coli endonuclease V corresponding to SEQ ID NO: 2. This mutantis generated by replacing the Tyrosine (Y) residue at the 75^(th)position of a wild type E. coli endonuclease V (SEQ ID NO: 1) with anAlanine (A) residue. In some embodiments, a mutant Afu endonuclease Y74A(SEQ ID NO: 4) and/or its conservative variants is employed. The mutantY74A Afu endonuclease is generated by substituting a Tyrosine (Y)residue at the 75^(th) position of a wild type Afu endonuclease V (SEQID NO: 3) with an alanine (A) residue.

Table 1 provides the sequences of wild type endonucleases and mutantendonuclease V enzymes.

TABLE 1Sequences of wild type endonucleases, mutant endonucleases, templateDNAs, and various primers Ref. No. Sequence (N-term-C-term; 5′→3′)Length Wide Type E. SEQ ID MIMDLASLRAQQIELASSVIREDRLDKD 223 coli NO: 1PPDLIAGADVGFEQGGEVTRAAMVLLK endonuclease V YPSLELVEYKVARIATTMPYIPGFLSFREYPALLAAWEMLSQKPDLVFVDGHGISH PRRLGVASHFGLLVDVPTIGVAKKRLCGKFEPLSSEPGALAPLMDKGEQLAWVWR SKARCNPLFIATGHRVSVDSALAWVQRCMKGYRLPEPTRWADAVASERPAFVRY TANQP Y75A mutant E. SEQ IDMIMDLASLRAQQIELASSVIREDRLDKD 225 coli NO: 2 PPDLIAGADVGFEQGGEVTRAAMVLLKendonuclease V YPSLELVEYKVARIATTMPAIPFELSFRE YPALLAAWEMLSQKPDLVFVDGHGISHPRRLGVASHFGLLVDVPTIGVAKKRLCG KFEPLSSEPGALAPLMDKGEQLAWVWRSKARCNPLFIATGHRVSVDSALAWVQR CMKGYRLPEPTRWADAVASERPAFVRY TANQPLEWild Type Afu SEQ ID MLQMNLEELRRIQEEMSRSVVLEDLIPL 221 endonuclease VNO: 3 EELEYVVGVDQAFISDEVVSCAVKLTFP ELEVVDKAVRVEKVTFPYIPTFLMFREGEPAVNAVKGLVDDRAAIMVDGSGIAHP RRCGLATYIALKLRKPTVGITKKRLFGEMVEVEDGLWRLLDGSETIGYALKSCRR CKPIFISPGSYISPDSALELTRKCLKGYKLPEPIRIADKLTKEVKRELTPTSKLK Y74A mutant SEQ IDMLQMNLEELRRIQEEMSRSVVLEDLIPL 221 Afu NO: 4 EELEYVVGVDQAFISDEVVSCAVKLTFPendonuclease V ELEVVDKAVRVEKVTFPAIPTELMFREG EPAVNAVKGLVDDRAAIMVDGSGIAHPRRCGLATYIALKLRKPTVGITKKRLFGE MVEVEDGLWRLLDGSETIGYALKSCRRCKPIFISPGSYISPDSALELTRKCLKGYKL PEPIRIADKLTKEVKRELTPTSKLK Wild Type TmaSEQ ID MDYRQLHRWDLPPEEAIKVQNELRKKI 225 endonuclease V NO: 5KLTPYEGEPEYVAGVDLSFPGKEEGLAV IVVLEYPSFKILEVVSERGEITFPYIPGLLAFREGPLFLKAWEKLRTKPDVVVFDGQ GLAHPRKLGIASHMGLFIEIPTIGVAKSRLYGTFKMPEDKRCSWSYLYDGEEIIGCV IRTKEGSAPIFVSPGHLMDVESSKRLIKAFTLPGRRIPEPTRLAHIYTQRLKKGLF Y80A mutant SEQ IDMDYRQLHRWDLPPEEAIKVQNELRKKI 225 Tma NO: 6 KLTPYEGEPEYVAGVDLSFPGKEEGLAVendonuclease V IVVLEYPSFKILEVVSERGEITFPAIPGLLAFREGPLFLKAWEKLRTKPDVVVFDGQ GLAHPRKLGIASHMGLFIEIPTIGVAKSRLYGTFKMPEDKRCSWSYLYDGEEIIGCV IRTKEGSAPIFVSPGHLMDVESSKRLIKAFTLPGRRIPEPTRLAHIYTQRLKKGLF

In some embodiments the method for producing at least one amplicon basedon a target DNA comprises the steps of (a) providing the target DNA; (b)providing a primer solution consisting essentially of anexonuclease-resistant, inosine-containing primer; (c) treating theprimer solution with an exonuclease to remove any contaminating nucleicacids from the primer solution; (d) inactivating the exonuclease in theprimer solution after the decontamination step (c); (e) generating a DNAamplification reaction mixture by mixing together the target DNA, thedecontaminated primer solution, at least one 5′→3′ exonuclease-deficientDNA polymerase having strand displacement activity, and at least oneendonuclease that is capable of nicking a DNA at a residue 3′ to aninosine residue; and (f) amplifying at least one portion of the targetDNA using the DNA amplification reaction mixture of step (e) to producethe at least one amplicon. The primer solution may further compriseother nucleic acid amplification reagents such as dNTPs andamplification buffers. The primer solution may further comprise one ormore of reagents such as formamide, SSB protein, ethylene glycol orFicoll. However, in embodiments wherein the primer solution consistsessentially of an exonuclease-resistant, inosine-containing primer theprimer solution, the primer solution is devoid of amplicons,exonuclease-resistant oligonucleotides, inosine-containingoligonucleotides, or exonuclease-resistant, inosine-containing primersthat are originated from previous amplification reactions (e.g., nucleicacid contaminants from prior amplification reactions).

The DNA amplification reaction mixture for nucleic acid amplificationreaction may further include one or more reagents such as surfactants(e.g., detergents), blocking reagents (e.g., albumin), topoisomerase,reducing agents or buffers. Generally, these reagents are added to theprimer solution, which contains the exonuclease-resistant,inosine-containing primer, before the decontamination step (e.g., priorto the treatment of the primer solution with an exonuclease to removeany contaminating nucleic acids). After the exonuclease treatment, thedecontaminated primer solution (that contains exonuclease-resistant,inosine-containing primer that is to be used for the DNA amplificationalong with other added reagents) is then used for generating the nucleicacid amplification reaction mixture (with or without the inactivation ofthe exonuclease employed for decontamination). In some specificembodiments, all the reagents that is required for DNA amplification(except the target DNA itself) is added to the primer solution prior tothe treatment of the primer solution with the exonuclease. In suchembodiments, almost all of the contaminated DNA (except the ones comingfrom the target DNA solution) may be removed from the DNA amplificationreaction mixture, thereby making target DNA amplificationcontamination-free.

Any buffers (e.g., Tris buffer, HEPES buffer) that results in a reactionpH between 6 and 9 may be used for DNA amplification reaction. In someembodiments, the pH of the nucleic acid amplification reaction mixtureis 7.7. In some embodiments, buffers that enhance DNA stability (e.g.,HEPES) may be used. Thermo labile buffers such as Tris-Borate, HEPES,and MOPS buffers may be disfavored in some specific DNA amplificationreactions that employ thermal denaturation of a target DNA or thermaldenaturation of the exonuclease after the decontamination step.Surfactants may be applied to the reaction tube before introducing thefirst component of the reaction mixture. Alternatively, as with otherreagents, surfactants may be added to the to the primer solution priorto the decontamination step by the exonuclease. In some embodiments, thesurfactant may be a detergent selected from Tween-20, NP-40,Triton-X-100, or combinations thereof. In some embodiments, 0.05% NP-40and 0.005% Triton X-100 is used for the reaction. In some specificembodiments, the DNA amplification reaction buffer may comprise 25 mMTris-Borate; 5 mM MgCl₂; 0.01% Tween; and 20% ethylene glycol. Blockingagents such as an albumin (e.g., BSA or HSA) may be included in the DNAamplification reaction mixture to bind to the surface of the reactionvessel (e.g., plastic microcentrifuge tube or microtiter plate) therebyincreasing the relative amount target DNA that is available for reactionwith the nucleases or polymerases. The DNA amplification reactionmixture may include at least one topoisomerase (e.g., a type 1topoisomerase). The topoisomerase may be present in the reaction mixtureat a final concentration of at least 0.1 ng/μL. In some embodiments, theDNA amplification reaction mixture may include at least one singlestranded DNA binding protein (e.g., E. coli SSB, T4 gene 32 protein (T4g32p), T7 gene 2.5 protein, Ncp7, recA, or combinations thereof). Thesingle stranded DNA binding protein may be present in the reactionmixture at a final concentration of at least 0.1 ng/μL. The DNAamplification reaction mixture may also include one or more reducingagents such as dithiothreitol (DTT), 2-mercaptoethanol ((3ME),Tris(carboxyethyl) phosphine (TCEP), or 2-mercaptoethylamine (MEA) thatreduces the oxidation of enzymes in the reaction mix and improves thequality and yield of the amplicons produced.

The amplicons produced by various embodiments of the present DNAamplification methods may be determined qualitatively or quantitativelyby any of the existing techniques. For example, for a qualitative orquantitative assay, terminal-phosphate-labeled ribonucleotides may beused in combination with a phosphatase during/after DNA amplificationreaction for color generation. In such embodiments, the terminalphosphate may be protected from dephosphorylation by usingterminal-phosphate methyl esters of dNTPs or deoxynucleosidetetraphosphates.

In some embodiments, the amplicon production kit comprises at least oneexonuclease-resistant, inosine-containing primer, at least one 5′→3′exonuclease-deficient DNA polymerase with strand displacement activity,and at least one endonuclease, which is capable of nicking DNA at aresidue 3′ to an inosine residue. The inosine residue of theinosine-containing primer is located at least 4 nucleotides downstreamof the 5′ terminal residue. In some embodiments, the inosine residue islocated at the 3′ penultimate position of the primer sequence. In someembodiments, the exonuclease-resistant, inosine-containing primercomprises at least one phosphorothioate linkage between the inosineresidue at the penultimate 3′ position and the 3′ terminal residue. Insome embodiments, the kit may further comprise reagents or a reagentsolution required for performing a DNA synthesis reaction. Theexonuclease-resistant, inosine-containing primer included in the kit mayeither be a specific primer, a partially random primer or a randomprimer. In some embodiments, the kit comprises multipleexonuclease-resistant, inosine-containing primers. In some embodimentsthe kit comprises at least one endonuclease V. The endonuclease V mayeither be a wild type endonuclease V or a mutant endonuclease V. In someembodiments, the kit comprises a Y75A mutant E. coli endonuclease V. TheDNA polymerase included in the kit may be a Klenow, 5′→3′exonuclease-deficient Bst DNA polymerase (the large fragment of Bst DNApolymerase), 5′→3′ exonuclease-deficient delta Tts DNA polymerase, exo(−) Klenow, or exo(−) T7 DNA polymerase (Sequenase™)

The kit may further comprise an SSB protein. Suitable SSB proteins thatmay be included in the kit include, but not limited to, but not limitedto, extreme thermostable single stranded DNA-binding protein (ET SSBfrom New England Biolabs, MA), rec A (e.g., E. coli RecA), Tth RecA(RecA homolog isolated from Thermus thermophilus from New EnglandBiolabs, MA), phage T4 gene-32 protein, T7 gene 2.5 protein, Ncp7, andE. coli SSB protein.

The kit may further comprise exonuclease that may be used todecontaminate the primer solution comprising the at least oneexonuclease-resistant, inosine-containing primer. Suitable exonucleasesthat the kit may comprise are, for example, but not limited to,exonuclease I, exonuclease III, exonuclease VII, T7 gene-6 exonuclease,spleen exonuclease, T5 D15 exonuclease and lambda exonuclease. In someembodiments, the kit comprises exonuclease III. In some otherembodiments, the kit may comprise a mixture of exonuclease I andexonuclease III. The combination of exonucleases may be provided in asingle vessel or in multiple vessels, packaged together. The kit mayfurther include an instruction manual detailing the specific componentsincluded in the kit and the protocols for using them in ade-contamination reaction or in a DNA amplification reaction or both.

Practice of the invention will be still more fully understood from thefollowing examples, which are presented herein for illustration only andshould not be construed as limiting the scope of the present inventionas defined by the appended claims.

EXAMPLES

Some abbreviations used in the examples section are expanded as follows:“mg”: milligrams; “ng”: nanograms; “pg”: picograms; “fg”: femtograms;“mL”: milliliters; “mg/mL”: milligrams per milliliter; “mM”: millimolar;“mmol”: millimoles; “pM”: picomolar; “pmol”: picomoles; “μ”:microliters; “min”: minutes and “h.”: hours.

All melting temperature values provided herein are determined accordingto the formula,100.5+(41*(yG+zC−16.4)/(wA+xT+yG+zC))−(820/(wA+xT+yG+zC))+16.6*LOG10([Na+]+[K+])−0.56(% EG)−0.32(% G)−0.62(% F) where w, x, y and z referto the number of adenosine, cytosine, guanosine and thymidine residues,respectively, contained in the primer, Na⁺ refers to the sodiumconcentration (mM), K⁺ refers to the potassium concentration (mM), EGrefers to the ethylene glycol concentration (%), G refers to theglycerol concentration (%), and F refers to the formamide concentration.

Tris-HCl and Tween 20 were obtained from Sigma Aldrich; dNTPs wereobtained from GE Healthcare; and NaCl was obtained from Ambion. Volumesshown in the following Tables are in microliters unless otherwiseindicated. Amplicons may be visualized and/or quantified using any ofart-recognized techniques (e.g., electrophoresis to separate species ina sample and observe using an intercalating dye such as ethidiumbromide, acridine orange, or proflavine). Amplicon production may alsobe tracked using optical methods (e.g., ABI Series 7500 Real-Time PCRmachine) and an intercalating dye (e.g., SYBR Green I). The ampliconsproduced in the following examples were visualized using electrophoresisor optical techniques.

HET Buffer is 10 mM HEPES Buffer, pH 8, 0.1 mM EDTA and 0.1% (v/v) Tween20. 10× denaturation buffer is 100 mM HEPES Buffer, pH 8.0, 1 mM EDTA,0.1% (v/v) Tween 20 and 10 mg/ml BSA. 10× Reaction Buffer is 150 mMHEPES Buffer, pH 8, 30 mM magnesium chloride, 1 mM manganese sulphate,2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 2.5 mM dTTP, 50 mM ammoniumsulphate, 10 mM TCEP and 0.1% (v/v) Tween 20. Enzyme Dilution Buffer is10 mM HEPES, pH 8, 1 mM TCEP, 0.5 mM EDTA, 0.01% (v/v) Tween 20 and 50%(v/v) glycerol. 5% (w/v) Ficoll 400 is equivalent to 5 g/100 ml ofwater.

Example 1 Endonuclease-Assisted Isothermal Amplification of a TemplateDNA

Endonuclease-assisted, isothermal amplification reaction has threereagent mixes that are prepared separately and then mixed together tocreate the final amplification reaction. These three reagent mixes areidentified as denaturation mix, enzyme mix and reaction mix.

The denaturation mix is prepared by combining 0.5 μl 10× denaturationbuffer, 1 μl of the desired target DNA template at the appropriateconcentration, 0.5 μl 25% (v/v) formamide, 1 μl of an appropriate oligomix containing nuclease-resistant primers and 2 μl of nuclease-freewater for a final volume of 5 μl. The denaturation mix is heated at 95°C. for two minutes and then placed at room temperature.

The enzyme mix is prepared by combining 0.34 μl of the Large fragment ofBst DNA polymerase (120 units/μl), 0.2 μl of E. coli SSB (5 μl/μl),0.041 μl of mutant E. coli endonuclease V (6.23 mg/ml) and 0.419 μl ofEnzyme Dilution Buffer for a final volume of 1 μl.

The reaction mix is prepared by combining 1 μl of 10× Reaction Buffer, 2μl ethylene glycol, 1 μl of 50% (v/v) Ficoll 400 and 1 μl of the Enzymemix for a final volume of 5 μl.

A complete Ping Pong reaction is assembled by pre-heating separatelyboth the Denaturation mix and Reaction mix at 45° C. for 30 seconds.Both of these mixes are then combined and incubated at 45° C. for onehour.

Following incubation the amplification is analyzed by gelelectrophoresis using a 15% Acrylamide TBE-Urea gel (Invitrogen)Immediately prior to gel loading, 3 μl of a completed Ping Pong reactionis combined with 6 μl Gel Loading Buffer II (Invitrogen) and heatdenatured at 95° C. for two minutes followed by immediate quenching onice. 5 μl of this heat-denatured Ping Pong reaction is then loaded inone well of the gel. Electrophoresis is accomplished according to themanufacturer's (Invitrogen) instructions. Once electrophoresis iscomplete, the gel is stained with a 20× solution of SYBR Gold(Invitrogen) for 15 minutes and then scanned for fluorescein with aTyphoon 9410 Variable Mode Imager (GE Healthcare).

Example 2 Endonuclease-Assisted Isothermal Amplification of a TemplateDNA Using Exonuclease-Resistant, Inosine-Containing Primers

The primers used in Endonuclease-assisted isothermal amplificationcontain an inosine as the penultimate 3′ base. Endonuclease V recognizesinosine as a non-natural base and nicks the DNA strand containing theinosine residue at one base 3′ to the lesion. This example demonstratesthe enhanced amplification kinetics using a nuclease-resistant primer,wherein the phosphate bond between the inosine reside and the terminal3′ base has been phosphorothioated.

Sequences for six forward and five reverse primers were identified inthe 5′ region of the Mycobacterium tuberculosis rpoB gene. Two sets ofthese 11 primers were synthesized, one set without phosphorothioationand the other set with phosphorothioation between the inosine and theterminal 3′ base of each primer. TABLE 2 and TABLE 3 provide thesequences of the various primers used in the examples.

TABLE 2 Non-phosphorothioated primers without phosphorothioation betweenthe inosine and the terminal 3′ base of each primer Primer Name Ref. No.Sequence (5′→3′) Length IA TBropB F1 SEQ ID NO: 6 ACAGCCGCTAGTCCTAIT 18IA TBropB F2 SEQ ID NO: 7 CCCGCAAAGTTCCTCIA 17 IA TBrpoB F3nSEQ ID NO: 8 ACCGGGTCTCCT TCIC 16 IA TBrpoB F4 SEQ ID NO: 9GCTGCGCGAACCACTTIA 18 IA TBrpoB F5 SEQ ID NO: 10 CCGTACCCGGAGCIC 15IA TBrpoB F6 SEQ ID NO: 11 CAGATTCCCGCCAGAIC 17 IA TBropB R2SEQ ID NO: 12 GGCGAACCGATCAIC 15 IA TBropB R3 SEQ ID NO: 13CGGCGGATTCGCIC 14 IA TBrpoB R4 SEQ ID NO: 14 GGTTGACATCACCCCIC 17IA TBrpoB R5 SEQ ID NO: 15 GAGCACCTCTTCCAGIC 17 IA TBrpoB R6SEQ ID NO: 16 CGATCGGAGACAGCTCIT 18

TABLE 3 Phosphorothioated primers with phosphorothioation(* represents phosphorothioate linkage) between the inosine and the terminal 3′ base of each primer.Primer Name Ref. No. Sequence (5′→3′) Length IA TBropB F1* SEQ ID NO: 17ACAGCCGCTAGTCCTA 18 I*T IA TBropB F2* SEQ ID NO: 18 CCCGCAAAGTTCCTC 17I*A IA TBrpoB F3n* SEQ ID NO: 19 ACCGGGTCTCCT TC 16 I*C IA TBrpoB F4*SEQ ID NO: 20 GCTGCGCGAACCACTT 18 I*A IA TBrpoB F5* SEQ ID NO: 21CCGTACCCGGAGCI*C 15 IA TBrpoB F6* SEQ ID NO: 22 CAGATTCCCGCCAGA 17 IC*IA TBropB R2* SEQ ID NO: 23 GGCGAACCGATCAI*C 15 IA TBropB R3*SEQ ID NO: 24 CGGCGGATTCGCI*C 14 IA TBrpoB R4* SEQ ID NO: 25GGTTGACATCACCCC 17 I*C IA TBrpoB R5* SEQ ID NO: 26 GAGCACCTCTTCCAG 17I*C IA TBrpoB R6* SEQ ID NO: 27 CGATCGGAGACAGCTC 18 I*T

To generate the non-phosphorothioated primer set, 4.00 μL IA TBrpoB F1(629 pmol/μL), 3.16 μL IA TBrpoB F2 (793 pmol/μL), 3.64 μL IA TBrpoB F3n(686 pmol/μL), 3.50 μL IA TBrpoB R2 (715 pmol/μL), 3.28 Ξl IA TBrpoB R3(762 pmol/μL), 5.74 μL IA TBrpoB F4 (436 pmol/L), 2.84 μL IA TBrpoB F5(880 pmol/μL), 4.17 μL IA TBrpoB F6 (599 pmol/μL), 4.94 μL IA TBrpoB R4(506 pmol/μL), 4.15 μL IA TBrpoB R5 (602 pmol/μL) and 5.63 μL IA TBrpoBR6 (444 pmol/μL) was mixed with 204.95 μL HE(0.1)T buffer (TotalVolume=250 μL)

To generate the phosphorothioated primer set, 3.32 μL IA TBrpoB F1* (754pmol/μL), 2.72 μL IA TBrpoB F2* (920 pmol/μL), 2.83 μL IA TBrpoB F3*(883 pmol/μL), 4.68 μL IA TBrpoB F4* (534 pmol/μL), 1.72 μL IA TBrpoBF5* (1451 pmol/μL), 1.76 μL IA TBrpoB F6* (1417 pmol/μL), 2.72 μL IATBrpoB R2* (920 pmol/μL), 1.80 μL IA TBrpoB R3* (1392 pmol/μL), 1.51 μLIA TBrpoB R4* (1652 pmol/μL), 1.79 μL IA TBrpoB R5* (1398 pmol/μl), and7.00 μL IA TBrpoB R6* (358 pmol/μL) was mixed with 218.15 μL HE(0.1)Tbuffer (Total Volume=250 μL)

Each primer set was then used in an endonuclease-assisted isothermalamplification reaction prepared and analyzed as in Example 1. The finalconcentration of each oligonucleotide primer in the Ping Pong reactionwas 10 pmol. As depicted in FIG. 3, use of nuclease-resistant primers(i.e., primer set containing phosphorothioation) increase the yield ofendonuclease-assisted isothermal amplification reaction products byabout a factor of two.

Example 3 Endonuclease-Assisted Isothermal Amplification of a TemplateDNA Using Contamination-Free Reagents

In this Example, all Ping Pong reagents, except, the DNA polymerase,mutant Endonuclease V and the human genomic DNA templates aredecontaminated to remove any exogenous DNA by a pre-amplificationincubation step with exonucleases. In this example embodiment, 11 ng ofhuman genomic DNA is used as the template in an amplification reactionand an exonuclease-resistant, inosine-containing oligonucleotides areused as the primers. For this example, exonuclease I (Exo I) andexonuclease III (Exo III) were first incubated with the appropriatereagents for a certain time interval and then killed by heating. Thecleaned reagents were then incorporated into a Ping Pong reaction.

A mixture containing 1.25 μL exonuclease I (New England Biolabs, 0.25Units/μL), 1 μL exonuclease III (New England Biolabs, 1 Unit/μL) and97.95 μL Enzyme Dilution Buffer was prepared. One microliter of thisexonuclease mixture was added to 0.5 μL Denaturation Buffer, 1 μL 10×Reaction Buffer, 0.5 μL 25% (v/v) formamide, 0.2 μL SSB, 0.419 μL EnzymeDilution Buffer, 2 μL ethylene glycol, 1 μL 50% (v/v) Ficoll 400, 1 μLof p53 Oligo Mix (phosphorothioated primers employed for p53amplification as listed in TABLE 2) and 1 μL water for a final volume of8.619 μL. The cleaning reaction was incubated at 37° C. for 30 minutesto allow the exonucleases to degrade any contaminating DNA and thenincubated at 80° C. for 30 minutes to deactivate the exonucleases. Thecleaned reagents were stored at +4° C. until required.

TABLE 4 Phosphorothioated primers (* representsphosphorothioate linkage) employed for p53 amplification. Primer NameRef. No. Sequence (5′→3′) Length IA p53 F1* SEQ ID NO: 28GCCTCGCCTCCCGAI*T 16 IA p53 F2* SEQ ID NO: 29 CTGGGATTACAGGCAT I*C 18IA p53 F4* SEQ ID NO: 30 CTCCCGGGTTCAAGCI*A 17 IA p53 F5* SEQ ID NO: 31GAGATCTCAGCTCACCI*C 18 IA p53 F6* SEQ ID NO: 32 CAGGCTGGAGTGTAAT I*G 18IA p53 F7* SEQ ID NO: 33 GACGGAGTTTCACTCTTI*T 19 IA p53 R2*SEQ ID NO: 34 CTGAGGTCGGGAGTTTI*A 18 IA p53 R3* SEQ ID NO: 35GAGGCCAAGGCGAGTI*I 17 IA p53 R4* SEQ ID NO: 36 GGCGCAGTGGCTCACI*A 17IA p53 R5* SEQ ID NO: 37 AAAATGGGGTAAGGGGI*C 18 IA p53 R6* SEQ ID NO: 38ACCCCCGTCAAACTCAI*T 18 IA p53 R7* SEQ ID NO: 39 GTCATATACTCAGCCCTI*C 19

To generate the phosphorothioated primer set for p53 gene segmentamplification, 5.96 μL IA p53 F1* (839 pmol/μL), 6.05 μL IA p53 F2* (826pmol/μL), 7.40 μL IA p53 F3* (676 pmol/μL), 6.55 μL IA p53 F4* (763pmol/μL), 10.99 μL IA p53 F5* (455 pmol/μL), 8.50 μL IA p53 F6* (588pmol/μL), 6.17 μL IA p53 R1* (811 pmol/μL), 11.24 μL IA p53 R2* (445pmol/μL), 15.38 μL IA p53 R3* (325 pmol/μL), 4.81 μL IA p53 R4* (1040pmol/μL), 4.68 μL IA p53 R5* (1069 pmol/μL), 8.05 μL IA p52 R6* (621pmol/μL), was mixed with 404.22 μL 0.01% Tween 20 (Total Volume=500 μL)

0.34 μL of the Large Fragment of Bst DNA polymerase and 0.041 μL ofmutant Endonuclease V were added to the cleaned reagents along witheither 1 μl (11 ng; ˜1500 copies) of human genomic DNA or water (notemplate controls (NTC)). Control reactions without cleaning wereprepared as in Example 2. All reactions were incubated and analyzed asin Example 2. FIG. 1 shows that cleaning of the Ping Pong reagentsincluding exonuclease-resistant inosine containing primer with anexonuclease eliminates non-specific amplification products.

The above detailed description is exemplary and not intended to limitthe invention of the application and uses of the invention. Throughoutthe specification, exemplification of specific terms should beconsidered as non-limiting examples. The singular forms “a”, “an” and“the” include plural referents unless the context clearly dictatesotherwise. Approximating language, as used herein throughout thespecification and claims, may be applied to modify any quantitativerepresentation that could permissibly vary without resulting in a changein the basic function to which it is related. Accordingly, a valuemodified by a term such as “about” is not to be limited to the precisevalue specified. Unless otherwise indicated, all numbers expressingquantities of ingredients, properties such as molecular weight, reactionconditions, so forth used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless indicated to the contrary, the numerical parametersset forth in the specification and attached claims are approximationsthat may vary depending upon the desired properties sought to beobtained by the present invention. At the very least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each numerical parameter should at least beconstrued in light of the number of reported significant digits and byapplying ordinary rounding techniques. Where necessary, ranges have beensupplied, and those ranges are inclusive of all sub-ranges therebetween.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are selected embodiments or examples from a manifold of allpossible embodiments or examples. The foregoing embodiments aretherefore to be considered in all respects as illustrative rather thanlimiting on the invention described herein. While only certain featuresof the invention have been illustrated and described herein, it is to beunderstood that one skilled in the art, given the benefit of thisdisclosure, will be able to identify, select, optimize or modifysuitable conditions/parameters for using the methods in accordance withthe principles of the present invention, suitable for these and othertypes of applications. The precise use, choice of reagents, choice ofvariables such as concentration, volume, incubation time, incubationtemperature, and the like may depend in large part on the particularapplication for which it is intended. It is, therefore, to be understoodthat the appended claims are intended to cover all modifications andchanges that fall within the true spirit of the present invention.Further, all changes that come within the meaning and range ofequivalency of the claims are intended to be embraced therein.

1. A method of producing at least one amplicon based on a target DNAcomprising: (a) providing the target DNA; (b) providing a primersolution comprising at least one exonuclease-resistant,inosine-containing primer; (c) generating a DNA amplification reactionmixture by mixing together the target DNA, the primer solution, at leastone 5′→3′ exonuclease-deficient DNA polymerase having a stranddisplacement activity, and at least one endonuclease that is capable ofnicking an inosine-containing strand of a double stranded DNA at aresidue 3′ to an inosine residue; and (c) incubating the DNAamplification reaction mixture to amplify at least one portion of thetarget DNA using the at least one exonuclease-resistant,inosine-containing primer to produce the at least one amplicon.
 2. Themethod claim 1, wherein the primer solution further comprises freedeoxynucleotide triphosphates (dNTPs).
 3. The method of claim 2, whereinthe primer solution further comprises DNA amplification buffer,formamide, single stranded DNA binding protein, ethylene glycol, Ficollor combinations thereof.
 4. The method of claim 1, claim 2, or claim 3further comprising decontaminating the primer solution, prior togenerating the DNA amplification reaction mixture, by treating theprimer solution with an exonuclease to remove any contaminating nucleicacids.
 5. The method of claim 4, further comprising inactivating theexonuclease in the primer solution after removal of any contaminatingnucleic acids prior to generating the DNA amplification reactionmixture.
 6. The method of claim 5, wherein the target DNA is amplifiedunder isothermal conditions.
 7. The method of claim 1, wherein theinosine residue of the nuclease-resistant, inosine-containing primer islocated at least 4 nucleotides downstream of the 5′ terminal nucleotide.8. The method of claim 7, wherein the inosine residue of thenuclease-resistant, inosine-containing primer is located at thepenultimate 3′ position.
 9. The method of claim 8, wherein thenuclease-resistant, inosine-containing primer comprises aphosphorothioate linkage at the 5′ side or at the 3′ side of the inosineresidue.
 10. The method of claim 1, wherein the inosine-containingprimer comprises at least 2 adjacent inosine residues.
 11. The method ofclaim 10, wherein the inosine residues are located both at thepenultimate 3′ position and the 3′ terminal end of theinosine-containing primer.
 12. The method of claim 1, wherein the atleast one 5′→3′ exonuclease-deficient DNA polymerase is selected from5′→3′ exonuclease-deficient T7 DNA polymerase, 5′→3′exonuclease-deficient Bst DNA polymerase, 5′→3′ exonuclease-deficientKlenow, 5′→3′ exonuclease-deficient delta Tts DNA polymerase, orcombinations thereof.
 13. The method of claim 1, wherein the primersolution comprises at least one inosine-containing forward primer and atleast one inosine-containing reverse primer, wherein both the forwardprimer and the reverse primer are exonuclease-resistant.
 14. The methodof claim 1, wherein the at least one nuclease-resistant,inosine-containing primer comprises an extender template.
 15. A methodfor producing at least one amplicon based on a target DNA comprising:(a) providing the target DNA; (b) providing a primer solution consistingessentially of an exonuclease-resistant, inosine-containing primer; (c)treating the primer solution with an exonuclease to remove anycontaminating nucleic acids from the primer solution; (d) inactivatingthe exonculease in the primer solution after step (b). (e) generating aDNA amplification reaction mixture by mixing together the target DNA,the primer solution of step (d), a 5′→3′ exonuclease-deficient DNApolymerase having strand displacement activity, and a endonuclease thatis capable of nicking a DNA at a residue 3′ to an inosine residue; and(f) amplifying at least one portion of the target DNA using theamplification reaction mixture of step (e) to produce the at least oneamplicon.
 16. The method claim 15, wherein the primer solution furthercomprises free nucleotides (dNTPs) and reagents slected from the groupconsisting of DNA amplification buffer, formamide, single stranded DNAbinding protein, ethylene glycol, Ficoll and combinations thereof.
 17. Akit for endonuclease-assisted isothermal nucleic acid amplification,comprising: at least one exonuclease-resistant, inosine-containingprimer; at least one 5′→3′ exonuclease-deficient DNA polymerase withstrand displacement activity; and at least one endonuclease, which iscapable of nicking DNA at a residue 3′ to an inosine residue.
 18. Thekit of claim 17, wherein the inosine residue of the inosine-containingprimer is located at least 4 nucleotides downstream of the 5′ terminalnucleotide.
 19. The kit of claim 18, wherein the inosine residue of theinosine-containing primer is located at the penultimate 3′ position. 20.The kit of claim 19, wherein the inosine-containing primer comprises atleast one phosphorothioate linkage between the inosine residue at thepenultimate 3′ position and the 3′ terminal residue.
 21. The kit ofclaim 20, wherein the at least one 5′→3′ exonuclease-deficient DNApolymerase is selected from 5′→3′ exonuclease-deficient T7 DNApolymerase, 5′→3′ exonuclease-deficient Bst DNA polymerase, 5′→3′exonuclease-deficient Klenow, 5′→3′ exonuclease-deficient delta Tts DNApolymerase, or combinations thereof.
 22. The kit of claim 21, whereinthe at least one nuclease is an endonuclease V.